The invention generally relates to a method for water-solubilization of cytotoxic trans-platinum compounds and to a method of killing tumor cells. In particular, the invention provides cytotoxic platinum compounds of the general formula SP-4-2-[PtX(L)(Lxe2x80x2)(B)]+for the treatment of tumors.
The use of cisplatin, cis-[PtCl2(NH3)2], and carboplatin, [Pt(CBDCA)(NH3)2] (CBDCA=1,1-cyclobutanedicarboxylate), in the treatment of certain cancers is well-established. Nevertheless, there is a continued interest in the design of structurally novel platinum compounds that show antitumor activity complementary to that of the clinical drugs. The fact that transplatin, trans-[PtCl2(NH3)2], 
was found to be therapeutically inactive, has been considered a paradigm for the structure-activity relationships (SAR) of platinum(II) antitumor compounds; trans-Pt compounds have been dismissed as ineffective in vivo agents.
However, the presence of a planar ligand such as pyridine or quinoline, e.g., in trans-[PtCl2 (NH3) (quinoline)], 
dramatically enhances the in vitro cytotoxicity of the trans geometry [Farrell, N., Kelland, L. R., Roberts, J. D. and Van Beusichem, M.: Activation of the Trans Geometry in Platinum Antitumor Complexes. A Survey of the Cytotoxicity of Trans Complexes Containing Planar Ligands in Murine L1210 and Human Tumor Panels and Studies on Their Mechanism of Action. Cancer Res. 52:5065 (1992); Van Beusichem, M. and Farrell, N.: Activation of the Trans Geometry in Platinum Antitumor Complexes. Synthesis, Characterisation and Biological Activity of Complexes with Planar Ligands Pyridine, N-Methylimidazole, Thiazole and Quinoline. The Crystal and Molecular Structure of trans-dichlorobis(thiazole)platinum(II). Inorg. Chem. 31:634 (1992)] The cytotoxic activity of such xe2x80x9cnonclassicalxe2x80x9d trans-platinum complexes has been discussed in terms of both an overall altered affinity toward biologically relevant (N and S) nucleophiles and unique DNA binding modes. Importantly, the newer trans-platinum compounds containing planar ligands display a different profile of cytotoxicity in comparison to cisplatin and retain their cytotoxic activity in cisplatin-resistant tumor cells. Thus, there is reason to believe that a trans-platinum compound in the clinic would have activity complementary to cisplatin, resulting in significant benefits to patients. However, such xe2x80x9cnonclassicalxe2x80x9d trans-platinum species have been found to have limited bioavailability and, consequently, low in vivo activity. One possible explanation is lack of water solubility.
It would be highly desirable to have available additional platinum species for the treatment of cancer. It would be especially desirable if such compounds displayed high levels of cytotoxicity and were also water-soluble, thereby enhancing their bioavailability and potential in vivo usefulness for the treatment of tumors.
It is an object of this invention to provide a method for enhancing water-solubility and cytotoxicity of the trans-platinum geometry through production of cationic compounds.
It is a further object of this invention to provide a method for killing tumor cells and treating tumors in patients, comprising the step of administering to a patient in need thereof an effective amount of a platinum coordination compound of the general formula SP-4-2-[PtX(L)(Lxe2x80x2)(B)]+ where X is an anionic ligand; L and Lxe2x80x2 represent ammines (NH3) or substituted or unsubstituted heterocyclic amines where the substituents are electrophilic or nucleophilic, and L and Lxe2x80x2 may be the same or different, and B is a sulfoxide, usually dimethylsulfoxide R2R3SO (where R2=methyl and R3=methyl; however it should be understood that other alkyl substituted sulfoxides may be used in this invention and that R2 and R3 may be the same or different) or a heterocyclic nucleobase with a nitrogen in a ring which is connected to Pt.
In preferred embodiments of the present invention, the platinum coordination compound is trans-[PtCl(Me2SO)(pyridine)2]+, or trans-[PtCl(9-ethylguanine)(NH3)2]+, or SP-4-2-[PtCl(9-ethylguanine)(NH3)(thiazole)]+, or SP-4-2-[PtCl(9-ethylguanine) (NH3)(benzothiazole)]+, or SP-4-2-[PtCl 9-ethylguanine)(NH3)(quinoline)]+, or SP-4-2-[PtCl(9-ethylguanine)(NH3)(isoquinoline)]+, or trans-[PtCl(9-ethylguanine) (4-picoline)2]+, or trans- [PtCl(1-methylcytosine)(NH3)2]+, or SP-4-2-[PtCl (1-methylcytosine)(NH3)(thiazole)],+ or SP-4-2-[PtCl(1-methylcytosine)(NH3)(quinoline)]+, or SP-4-2-[PtCl(1-methylcytosine)(NH3)(isoquinoline)]+. Administration may be oral or parenteral.
It is a further object of the instant invention to provide new compositions of matter in the form of platinum coordination compounds: trans-[PtCl(Me2SO)(pyridine)2]+, SP-4-2-[PtCl(9-ethylguanine)(NH3)(thiazole)]+, SP-4-2-[PtCl(9-ethylguanine)(NH3) (benzothiazole)]+, SP-4-2-[PtCl(9-ethylguanine)(NH3)(isoquinoline)]+, trans-[PtCl (9-ethylguanine)(4-picoline)2]+, trans-[PtCl(1-methylcytosine)(NH3)2]+, SP-4-2-[PtCl(1-methylcytosine)(NH3)(thiazole)],+ SP-4-2-[PtCl (1-methylcytosine) (quinoline)]+, and SP-4-2-[PtCl(1-methylcytosine)(NH3)(isoquinoline)]+.
Platinum compounds of the general formula [PtXAmB3xe2x88x92m], which show excellent solubility, have been previously described as anti-viral agents (U.S. Pat. No. 6,113,934, the complete contents of which is herein incorporated by reference). Surprisingly, some of these compounds also display cytotoxic properties. Therefore, the present invention provides novel forms of such compounds, a method for water-solubilization of cytotoxic trans-platinum compounds, a method for the use of such compounds as cytotoxic agents, and a method of use of such compounds to treat tumors. These compounds are prepared as salts, the cationic component of which is described by the formula SP-4-2[PtX(L)(Lxe2x80x2)(B)]+.
It is an object of the present invention to provide a method of killing tumor cells and treating tumors by the administration of platinum complexes of the general formula:
SP-4-2[PtX(L)(Lxe2x80x2)(B)]+
In this formula:
a) X represents an anionic ligand such as halogens (e.g., Cl, Br, or I), alkoxides (e.g., OR where Rxe2x95x90CH3, C2H5, or other lower alkyls), sulfhydryls (SR where Rxe2x95x90CH3, C2H5, or other lower alkyls, where C1-12 is preferred), and carboxylates (RCOOxe2x88x92 where Rxe2x95x90CH3, C2H5, etc.).Chloride is the preferred anionic ligand;
b) L and Lxe2x80x2 represent ammines (NH3 linked directly to the platinum metal), or substituted or unsubstituted heterocyclic amines, where the substituents are electrophilic or nucleophilic (e.g. C1-12 alkyl, xe2x80x94NO2, xe2x80x94X (Cl, Br, I), -NR2 where R is C1-12 alkyl, -COOR where R is C1-12 alkyl). Preferred heterocycles include but are not limited to thiazole, benzothiazole, imidazole, quinoline, isoquinoline, and picoline. Other useful heterocycles may include oxazole, indole, and acridine. L and Lxe2x80x2 may be the same of different. Further, L and Lxe2x80x2 are located xe2x80x9ctransxe2x80x9d to one another in the compounds. Note that, because four different substituents are present, xe2x80x9ccis-transxe2x80x9d designations technically do not apply. The nomenclature xe2x80x9cSP-4-2xe2x80x9d follows the rules from Nomenclature of Inorganic Chemistry, Recommendations 1990, Blackwell Publicaitons, 1990. Edited by G. J. Leigh. [ISBN 0-632-02319-8; 0-6323-02494-1 ]. This nomenclature indicates that the compounds are square planar Pt II compounds in which the two centrally named substituents, L and Lxe2x80x2, are located trans to each other as depicted below: 
c) B represents either of (i) or (ii):
i) a sulfoxide, R2R3 SO where R 2and R3 may be the same or different and represent alkyl or aryl substituents. In a preferred embodiment of the present invention, the sulfoxide is dimethylsulfoxide (where R2=methyl and R3=methyl). In yet another embodiment, R2=methyl and R3=benzyl;
ii) a nitrogen-containing nucleobase where the nitrogen is connected to the Pt moiety. Examples of such nucleobases include but are not limited to: purines and purine compounds (e.g. guanine, 9-ethylguanosine, adenine, hypoxanthine, xanthine, uric acid, caffeine, threobromine, and the like); pyrimidines and pyrimidine compounds (e.g. uracil, thymine, cytosine, methylcytosine, and the like); nucleosides (e.g. guanosine and the like); nucleotides (e.g. 5xe2x80x2-guanosinemonophosphate and the like); and oligonucleotides or defined polynucleotide sequences [e.g. deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptidonucleice acids (PNA)]. The preferred binding site of endocyclic nitrogens is N7 for purines and N3 for pyrimidines. Other ligands similar to nucleobases may also be used, for example, hydantoin.
The compounds in the present invention are usually cations and are prepared as salts. In depictions of the formulas in the text of the application, the counterion is omitted for simplicity. In preferred embodiments of the instant invention, the platinum compounds are prepared as nitrate salts. Other common counter-anions which may be utilized in the practice of the present invention include but are not limited to Clxe2x88x92, ClO4xe2x88x92, PF6xe2x88x92, and BF4xe2x88x92.
SP-4-2-[PtCl(nucleobase)(L)(Lxe2x80x2)]NO3. These compounds were prepared according to the published method (Bierbach U, Farrell N (1998) JBIC 3: 570-580).
To a solution of:
i) 1 mmol of trans-[PtCl2(L)(Lxe2x80x2)] (wherein L=Lxe2x80x2=NH3, or L=Lxe2x80x2=pyridine), or
ii) trans-[PtCl2(NH3)(L)] where L=pyridine or a planar amine as above; in 25 ml of anhydrous dimethylformamide (DMF) was added 0.170 g (1 mmol) of AgNO3. After stirring this mixture at room temperature in the dark for 48 hours, the precipitated AgCl was filtered off through a Celite pad. To the filtrate was added (1 mmol) of 9-ethylguanine (or other suitable nucleobase) and the mixture was allowed to stir for 48 hours. The DMF was removed under reduced pressure at 30xc2x0 C. After addition of 50 mL of diethyl ether, the remaining oil solidified. The obtained crude products was recrystallized from methanol or cold water. Identity of products was confirmed by NMR spectroscopy and elemental analysis.
SP-4-2-[PtCl(R2R3 SO)(L)(Lxe2x80x2)]NO3. These compounds contain a sulfoxide as ligand B. They were prepared in basically the same manner with slight modifications in work-up and crystallisation. The preparation is exemplified for the Me2SO case.
trans-[PtCl(Me2SO)(py)2]NO3 To a suspension of trans-[PtCl2(py)2] (1.0 g, 2.4 mmol) in 30 mL of MeOH was added AgNO3 (0.4 g, 2.4 mmol) and Me2SO (2 mL, 2.2 g, 28 mmol). The reaction mixture was stirred at 80xc2x0 C. overnight. The insoluble AgCl precipitate was filtered off and the filtrate was evaporated down. To the oil was added 2 ml of MeOH when a white solid precipitated out after stirring for about 10 minutes. Ether was then added to intensify the precipitation. After cooling overnight, the white solid was filtered off and recrystallized from hot MeOH/ether. The product was dried in vacuum with heat. Yield 66 %. Anal. Calcd. for C12H16CIN3O4SPt: C, 27.25; H, 3.03; N, 7.95. Found C, 26.71; H, 2.95; N 7.56.
trans-[PtCl (MeBzSO)(Py)2]NO3 The same general conditions were used as for the previous complex but with two equivalents of sulfoxide ligand. Upon evaporation to an oil, acetone was added to dissolve the excess of MeBzSO and the product was precipitated out with ether. Upon cooling, the white solid was filtered off, recrystallized from MeOH/ether and washed with acetone to remove any remaining free ligand. The product was dried in vacuum with heat. Yield 45 % Anal. Calcd. for C18H20CIN3O4SPt: C, 35.73; H, 3.31; N, 6.95. Found C, 35.93; H, 3.11; N, 6.70.
trans-[PtCl(Me2SO)(pic)2]NO3. The same general reaction conditions were used as above. Yield 68 % Anal. Calcd. for C14H20CIN3O4SPt: C, 30.19; H, 3.59; N, 7.55. Found C, 30.51; H, 3.94; N, 7.53.
trans-[PtCl (MeBzSO)(pic)2]NO3. The same general conditions were used as above but again with two equivalents of sulfoxide ligand. Upon evaporation to an oil the product was precipitated out with ether. After cooling overnight, the white solid was filtered off, recrystallized from hot MeOH/ether and washed with acetone to remove the excess of free ligand. The product was dried in vacuum with heat. Yield 53 % Anal. Calcd. for C19H22CIN3O4SPt: C, 37.94; H, 3.79; N, 6.64. Found C, 38.08; H, 3.73; N, 6.63.
Implementation of the claimed invention will generally involve identifying patients suffering from tumors and administering the platinum coordination compound in an acceptable form by an appropriate route. The dosage to be administered is usually determined in Phase I clinical trials and may vary depending on the age, gender, weight and overall health status of the individual patient, as well as the nature of the cancer itself.
Administration can be oral or parenteral, including intravenously, intramuscularly, subcutaneously, etc., or by other routes (e.g. transdermal, sublingual, aerosol, etc.).
The compounds can be administered in the pure form or in a pharmaceutically acceptable formulation including suitable elixirs, binders, and the like or as pharmaceutically acceptable salts or other derivatives. It should be understood that the pharmaceutically acceptable formulations and salts include liquid and solid materials conventionally utilized to prepare injectable dosage forms and solid dosage forms such as tablets and capsules. Water may be used for the preparation of injectable compositions which may also include conventional buffers and agents to render the injectable composition isotonic. Other potential additives include: colorants; surfactants (TWEEN, oleic acid, etc.); and binders or encapsulants (lactose, liposomes, etc). Solid diluents and excipients include lactose, starch, conventional disintergrating agents, coatings and the like. Preservatives such as methyl paraben or benzalkium chloride may also be used. Depending on the formulation, it is expected that the active composition will consist of 1-99% of the composition and the vehicular xe2x80x9ccarrierxe2x80x9d will constitute 1-99% of the composition. The pharmaceutical compositions of the present invention may include any suitable pharmaceutically acceptable additives or adjuncts to the extent that they do not hinder or interfere with the therapeutic effect desired of the Pt complex.
The administration of pharmaceutical compositions of the present invention can be intermittent, or at a gradual or continuous, constant or controlled rate to a patient. In addition, the time of day and the number of times per day that the pharmaceutical formulation is administered can vary. Further, the effective dose can vary depending upon factors such as the mode of delivery, gender, age, and other conditions of the patient, as well as tumor type and stage or grade.
Generally, for parenteral administration in humans, dosages in the range of from about 0.1 to about 500 mg active Pt compound/kg body weight/24 hr., more preferably 1.0 to 10.0 active Pt compound/kg body weight/24 hr., are effective. The level of efficacy and optimal amount of dosage for any given Pt complex may vary from complex to complex.